In a few bacterial and mammalian selenoenzymes the selenium, present as a selenocysteine residue in the protein, serves as a redox center.However, selenocysteine in the selenoprotein A component of clostridial glycine reductase has a unique biological role. A carboxymethyl group derived from the carbon skeleton of glycine is transferred to the selenium and this protein-bound selenoether intermediate is then reductively cleaved to form a thiolester derivative of a third protein component, protein C. Ibis ester is converted to acetylphosphate which serves as a source of ATP. The Se-carboxymethyl derivative of reduced selenoprotein A was prepared directly by reaction with [14C]bromoacetate. Time, temperature, and pH conditions that allowed complete reaction with the selenocysteine residue but limited formation of carboxymethylcysteine residues in the protein were determined. When incubated with protein C in the presence of arsenate, the radioactive Se-carboxymethyl group was converted to [14C]acetate. This conversion was inhibited if a cysteine residue on a selenoprotein A molecule also had reacted with the alkylating agent. Procedures for larger scale isolation of protein C were developed and a number of properties of this unusual enzyme were determined. The protein, M.W. about 200,000, consists of two dissimilar subunits of 90,000 and 110,000, which are dimers of 48,000 and 59,000 M. W. subunits, respectively. Non-linear increases in enzyme activity observed as a function of protein concentration, typical of associating-disassociating systems, are observed with protein C. Linearity can be restored by addition of alkylated protein C which alone is catalytically inactive. Isolation of the active factor is in progress. Studies with substrate amounts of enzyme are designed to elucidate the mechanism of the novel conversion of the carboxymethyl selenoether to an acetyl thiolester.